1. Field of the Invention
The present invention relates generally to the fields of protein biochemistry and fluorescence (Förster) resonance energy transfer. More specifically, the invention relates to the use of polarized/depolarized light to measure FRET.
2. Description of Related Art
Measurement of intracellular processes and protein-protein interactions in living cells relies extensively on green fluorescent protein-based bioassays. Many of these assays incorporate measurement of Förster resonance energy transfer (FRET) between different colors of fluorescent proteins, most commonly, the cyan (CFP) and yellow fluorescent proteins (YFP) (Miyawaki, 2003). Given the inherent sensitivity of FRET measurements to distance and orientation (Förster, 1948; Clegg, 1992), the changes in energy transfer are difficult to measure between FRET partners with broadly overlapping spectra, such as fluorescent proteins. Crosstalk excitation of the donor YFPs also leads to “false positive” indication of FRET, and serves as a barrier for adaptation of CFP:YFP based assays to high throughput assays. Although numerous corrective algorithms and methods have been developed to handle this problem (Jares-Erijman and Jovin, 2003), these methods are generally difficult to apply and can introduce additional error into the measurement of FRET. These methods also require extensive controls, which gives rise to additional data-handling and storage issues when adapted to a high-throughput approach. Thus, an optimal solution would require collection of a minimal set of images and corrective processing to determine rigorously the presence or absence of FRET.